Synthesis and turnover of pancreatic islet proinsulin and insulin will be determined in vivo in normal and diabetic animals, under defined nutritional, hormonal, and drug induced states. The in vivo determinations will be made by injecting H3- and C14-amino acids, purifying the hormones by immunochemical means, and measuring the rate of change of the specific activities of the hormones with time. These measurements will be compared with in vitro determinations on isolated islets, and cells in culture. Diabetic mutant mice will be similarly studied to identify defects in genetic regulation of insulin biosynthesis. Proinsulin messenger RNA has been extracted from fish islets, and will be purified. Its translation product will be analyzed to determine whether a precursor exists. The structure of the mRNA will be determined with respect to total nucleotides, base composition, poly-A content, and nontranslated codons. Purified proinsulin mRNA will be used with RNA-dependent DNA polymerase to synthesize high specific activity complementary DNA (cDNA). The proinsulin cDNA will be used by nucleic acid hybridization techniques to study regulation of proinsulin genes in development and in control of proinsulin synthesis. The cDNA may also be used as an affinity column for isolation of the proinsulin gene. Studies will be performed on isolated rat islets to determine the mechanisms whereby alterations of glucose concentrations regulate translation of proinsulin mRNA. Inhibitors of polypeptide initiation will be used to determine their effects on proinsulin synthesis. Efforts will be made to detect a proinsulin precursor in secretion granules, and in nascent chains from proinsulin synthesizing polyribosomes by immunoprecipitation.